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. 2018 May 5;8(9):e2842.
doi: 10.21769/BioProtoc.2842.

Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1

Prarthana Mohanraju  1 , John Van Der Oost  1 , Martin Jinek  2 , Daan C Swarts  2
Affiliations

Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1

Prarthana Mohanraju et al. Bio Protoc. .

Abstract

This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments. Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli.

Keywords: CRISPR-Cas; Cas12a; Cpf1; Protein purification.

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Figures

Figure 1.
Figure 1.. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli
Figure 2.
Figure 2.. Schematic of the Cas12a crRNA-DNA-targeting complex.
The expected cleavage sites are indicated by red arrows.
Figure 3.
Figure 3.. After lysis, the cell suspension becomes tinted brown and less viscous
Figure 4.
Figure 4.. Representative elution chromatogram of FnCas12a purified by Histrap HP (5 ml) affinity purification.
75 ml of cell-free extract was loaded on the column. Elution fractions were 1 ml and the flow rate was set at 2 ml/min. Absorbance at 280 nm is expressed in milli-absorbance units for the A280 (blue) and A254 (red). Please note that the A254 is not very informative after niNTA purification, as at this stage, the sample is contaminated with various nucleic acids. The green line indicates the concentration of Elution Buffer (0% at the start of the chromatogram and 100% at the final stage of the chromatogram).
Figure 5.
Figure 5.. Representative elution chromatogram of FnCas12a purified by Heparin FF (5 ml) affinity purification.
50 ml of sample was loaded. The flow rate was set at 2 ml/min and elution fractions were 1 ml. Absorbance is expressed in milli-absorbance units for the A280 (blue) and A254 (red). The green line indicates the concentration of IEX-B (0% at the start of the chromatogram with the concentration raising to 50% over 60 ml, and at 100% at the final stage of the chromatogram to wash the column).
Figure 6.
Figure 6.. Representative Coomassie Brilliant Blue stained 12% SDS-PAGE gel on which FnCas12a Heparin FF elution fractions were resolved.
M: PageRulerTM Plus Prestained Protein Ladder. Marker band sizes are indicated in kDa. IN: concentrated input sample of TEV protease-treated Histrap HP elution fractions after overnight dialysis. FT: Flow through from the column. Besides the large band formed by FnCas12a, other (contamination) bands can be observed. A9-C1: Elution fractions near the protein absorbance peak. The FnCas12a protein appears as a band with a size slightly larger than 130 kDa. In this case, fractions B14-C1 were combined.
Figure 7.
Figure 7.. Representative elution chromatogram of FnCas12a resolved on a HiLoad 16/600 Superdex 200 pg column.
1 ml of sample was loaded. Elution fractions were 1 ml and the flow rate was set at 1 ml/min. Absorbance is expressed in milli-absorbance units for the A280 (blue) and A254 (red).
Figure 8.
Figure 8.. Representative Coomassie Brilliant Blue stained 12% SDS-PAGE gel on which FnCas12a SEC elution fractions were resolved.
M: PageRuler Plus Prestained Protein Ladder. Marker band sizes are indicated in kDa. IN: concentrated input sample. Besides the large band formed by FnCas12a, other (contamination) bands can be observed. C1-C12: Elution fractions near the protein absorbance peak. The FnCas12a protein appears as a band with a size slightly larger than 130 kDa. In this case, fractions C3-C9 were combined.
Figure 9.
Figure 9.. A 2,774 bp linear target DNA substrate is cleaved by the FnCas12:crRNA complex, yielding products of 1,209 bp and 1,565 bp.
M: GeneRuler 1 kb DNA Ladder.
See this image and copyright information in PMC

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